Showing papers on "Xylanase published in 1994"
Patent•
02 Mar 1994TL;DR: An enzyme exhibiting xylanase activity, which enzyme is immunologically reactive with an antibody raised against a purified Xylanase derived from Aspergillus aculeatus, CBS 101.43 as mentioned in this paper.
Abstract: An enzyme exhibiting xylanase activity, which enzyme is immunologically reactive with an antibody raised against a purified xylanase derived from Aspergillus aculeatus, CBS 101.43. The enzyme may be used for degrading plant cell wall components e.g. in the preparation of feed, in baking, in the paper and pulp industry and in connection with separation of wheat into starch and gluten.
192 citations
TL;DR: The thermostability of the 20 396 Da Bacillus circulans xylanase was increased by the introduction of both intra- and intermolecular disulfide bridges by site-directed mutagenesis; in one case, to obtain favourable geometry additional mutations around the cysteine sites were designed by computer modelling.
Abstract: The thermostability of the 20 396 Da Bacillus circulans xylanase was increased by the introduction of both intra- and intermolecular disulfide bridges by site-directed mutagenesis. Based on the 3-D structure of the enzyme, sites were chosen where favourable geometry for a bridge existed; in one case, to obtain favourable geometry additional mutations around the cysteine sites were designed by computer modelling. The disulfide bonds introduced into the xylanase were mostly buried and, in the absence of protein denaturants, relatively insensitive to reduction by dithiothreitol. The mutant proteins were examined for residual enzymatic activity after various thermal treatments, and were assayed for enzymatic activity at elevated temperatures to assess their productivity. We have examined one of these mutants by X-ray crystallography. All of the disulfide bond designs tested increased the thermostability of the B. circulans xylanase, but not all enhanced the activity of the enzyme at elevated temperatures.
154 citations
TL;DR: Domains of this type are not restricted to cellulases, but are widely distributed between hemicellutases also, and therefore play a pivotal role in the activity of the whole repertoire of plant cell‐wall hydrolases.
Abstract: Summary
Cellulases expressed by Cellulomonas fimi consist of a catalytic domain and a discrete non-catalytic cellulose-binding domain (CBD). To establish whether CBDs are common features of plant cell-wall hydroiases from C. fimi, the molecular architecture of xylanase D (XYLD) from this bacterium was investigated. The gene encoding XYLD, designated xynD, consisted of an open reading frame of 1936 bp encoding a protein of Mr 68000. The deduced primary sequence of XYLD was confirmed by the size (64kDa) and N-terminal sequence of the purified recombinant xylanase. Biochemical analysis of the purified enzyme revealed that XYLD is an endo-acting xylanase which displays no detectable activity against polysaccharides other than xylan. The predicted primary structure of XYLD comprised an /V-terminal signal peptide followed by a 190-residue domain that exhibited significant homology to Family-G xylanases. Truncated derivatives of xynD, encoding the W-terminal 193 amino acids of mature XYLD directed the synthesis of a functional xylanase, confirming that the 190-residue N-terminal sequence constitutes the catalytic domain. The remainder of the enzyme consisted of two approximately 90-residue domains, which exhibited extensive homology with each other, and limited sequence identity with CBDs from other polysaccharide hydrolases. Between the two putative CBDs is a 197-amino-acid sequence that exhibits substantial homology with Rhizobium NodB proteins. The four discrete domains in XYLD were separated by either threonine/prolineor novel glycine-rich linker regions. Although full-length XYLD adsorbed to cellulose, truncated derivatives of the enzyme lacking the C-terminal CBD hydrolysed xylan but did not bind to cellulose. Fusion of the C-terminal domain to glutathione-Stransferase generated hybrid proteins that bound to crystalline cellulose, but not to amorphous cellulose or xylan. The location of CBDs in a C. fimi xylanase indicates that domains of this type are not restricted to cellulases, but are widely distributed between hemicellutases also, and therefore play a pivotal role in the activity of the whole repertoire of plant cell-wall hydrolases. The role of the NodB homologue in XYLD is less certain.
107 citations
TL;DR: Glu78 is completely conserved in all members of this xylanase family and indeed is shown to be located in the active site in the recently determined X-ray crystal structure.
Abstract: A new mechanism-based inactivator of beta-1,4-xylanases, 2',4'-dinitrophenyl 2-deoxy-2-fluoro-beta-xylobioside, has been synthesized and used to trap the covalent intermediate formed during catalysis by Bacillus subtilis xylanase. Electrospray mass spectrometry confirmed the 1:1 stoichiometry of the incorporation of inactivator into the enzyme. Inactivation of xylanase followed the expected pseudo-first-order kinetic behavior, and kinetic parameters were determined. The intermediate trapped was relatively stable toward hydrolytic turnover (t1/2 = 350 min). However, turnover could be facilitated by transglycosylation following the addition of the acceptor benzyl thio-beta-xylobioside, thus demonstrating the catalytic competence of the trapped intermediate. Reactivation kinetic parameters for this process of kre = 0.03 min-1 and Kre = 46 mM were determined. The nucleophilic amino acid was identified as Glu78 by a tandem mass spectrometric technique which does not require the use of radiolabels. The peptic digest of the labeled enzyme was separated by high-performance liquid chromatography and the eluent fed into a tandem mass spectrometer via an electrospray ionization device. The labeled peptide was identified as one of m/z = 826 (doubly charged) which fragmented in the collision chamber between the mass analyzers with loss of the mass of a 2-fluoroxylobiosyl unit. Confirmation of the peptide identity was obtained both by tandem mass spectrometric sequencing and by Edman degradation of the purified peptide. Glu78 is completely conserved in all members of this xylanase family and indeed is shown to be located in the active site in the recently determined X-ray crystal structure.
107 citations
TL;DR: Both xylanases displayed remarkable pH and thermal stabilities by retaining most of their activities even after having been subjected to temperatures much higher than their optimal, and remained active under prolonged storage, having no loss of activity after 1 month of storage at 4°C and retaining up to about 90% after 10 days at 55°C.
88 citations
TL;DR: Electrolytic and hemicellulolytic enzymes were produced on extracted sweet sorghum silage by mixed culture solid substrate fermentation with Trichoderma reesei LM-1 (a Peruvian mutant) and Aspergillus niger ATCC 10864.
Abstract: Cellulolytic and hemicellulolytic enzymes were produced on extracted sweet sorghum silage by mixed culture solid substrate fermentation with Trichoderma reesei LM-1 (a Peruvian mutant) and Aspergillus niger ATCC 10864. Optimal cellulose and xylanase levels of 4 IU/g dry weight (DW) and 180 IU/g DW, respectively, were achieved in 120 h-fermentation when T. reesei, inoculated at 0 h, was followed by the inoculation of A. niger at 48 h.
86 citations
TL;DR: Of the three endo-β-(1,4)-xylanases secreted by A. nidulans, that of 24 kDa was not under carbon catabolite repression, whereas the other two, of 22 and 34 kDa, were under glucose repression mediated by the creA gene product.
Abstract: The induction of the synthesis of extracellular xylanases was investigated in the fungus Aspergillus nidulans using a number of compounds, including xylans of different origin, monosaccharides, xylooligosaccharides and xylose derivatives. Certain xylans (wheat arabinoxylan, oat spelt xylan, birchwood xylan and 4-O-methyl-D-glucurono-D-xylan) were found to be the most powerful inducers. Also, xylooligosaccharides such as xylobiose, xylotriose and xylotetraose served as inducers, their efficiency being directly related to their chain length. Xylose, on the contrary, was not a true inducer. Of the three endo-β-(1,4)-xylanases secreted by A. nidulans, that of 24 kDa was not under carbon catabolite repression, whereas the other two, of 22 and 34 kDa, were under glucose repression mediated by the creA gene product.
84 citations
TL;DR: In this article, commercial xylanases, Novozyme 473, VAI Xylanase and Cartazyme HS-10 were evaluated for prebleaching of eucalyptus kraft pulp.
80 citations
TL;DR: An acidic xylanase from a culture filtrate of Aspergillus nidulans grown on oat-spelt xylan was purified to apparent homogeneity and the regulation of its synthesis has been studied.
Abstract: An acidic xylanase from a culture filtrate of Aspergillus nidulans grown on oat-spelt xylan was purified to apparent homogeneity. The purified enzyme showed a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis with a molecular mass of 34,000 Da and had an isoelectric point of approximately 3.4. The enzyme was a non-debranching endoxylanase highly specific for xylans. The xylanase showed an optimal activity at pH 6.0 and 56° C and had a Michaelis constant Km of 0.97 mg oat-spelt xylan (soluble fraction) ml and a maximed reaction velocity (Vmax) of 1,091 μmol min−1 (mg−1protein)−1. Using polyclonal antibodies raised against the purified enzyme, the regulation of its synthesis has been studied. The xylanase production is repressed by glucose and induced by oat-spelt xylan, arabinoxylan, 4-O-methylglucurono-xylan, birchwood xylan and xylose.
79 citations
TL;DR: It was found that the peptone medium with xylan (2.0%) is more appropriate to achieve high productivity of xylanase in bioreactor system, due to rheological problem under higher biomass formation with accompanying viscosity and consequent oxygen transfer efficiency.
79 citations
TL;DR: The esterases had only a small capacity to release ferulic and p -coumaric acids from cell walls of various plants, but they were more effective when acting in concert with the commercially available plant cell wall-degrading enzyme Celluclast.
TL;DR: Findings suggest that xylanase V is a novel enzyme that cleaves a xylobiose unit from one of the ends of xylans, probably by an exomechanism.
Abstract: A xylanase, which produces exclusively xylobiose from oat spelt and birch xylans, was isolated from the culture medium of Aeromonas caviae ME-1. The enzyme (xylanase V) was purified by ammonium sulfate fractionation, hydrophobic interaction, and ion-exchange and gel filtration chromatographies. The homogeneity of the final preparation was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and agarose gel electrofocusing. The molecular mass and isoelectric point of the xylanase were 46 kDa and 5.4, respectively. Xylanase V had a maximum activity at a pH of 6.8 and at a temperature between 30 and 37°C. It was relatively stable at a pH between 5.0 and 8.6 and a temperature between 25 and 37°C. When soluble birch xylan was used as the substrate, the enzyme had a Km and Vmax of 2 mg/ml and 182 μmol of xylose equivalent liberated · min-1 · mg of protein-1, respectively. By the action of xylanase V on xylans (from oat spelt and birch), only one product corresponding to xylobiose was observed by thin-layer chromatography. The xylanase V putative product was confirmed to be xylobiose by acid and enzymatic hydrolyses. The xylanase had neither β-xylosidase, α-l-arabinofuranosidase, cellulase, nor β-1,3-xylanase activities. Xylotriose was the shortest substrate which the enzyme could attack. These findings suggest that xylanase V is a novel enzyme that cleaves a xylobiose unit from one of the ends of xylans, probably by an exomechanism. Images
TL;DR: Alkaliphilic and thermophilic Bacillus sp.
Abstract: Alkaliphilic and thermophilic Bacillus sp. strain TAR-1, isolated from soil, produced a xylanase extracellularly. The xylanase was most active over a pH range of 5.0 to 9.5 at 50°C. Optimum temperatures of the crude xylanase preparation were 75°C at pH 7.0 and 70°C at pH 9.0. Zymogram analyses of the culture supernatant showed that the molecular mass of the xylanase was 40 kDa and the isoelectric point was pH 4.1. The predominant products of xylan hydrolysate were xylobiose, xylotriose, and higher oligosaccharides, indicating that the enzyme was an endoxylanase. Production of the thermophilic alkaline xylanase was induced by xylan and xylose, but was repressed in the presence of glucose.
TL;DR: It is proposed that the C. papyrosolvens enzyme system responsible for the hydrolysis of crystalline cellulose and xylan is a multicomplex system comprising at least seven diverse protein complexes.
Abstract: The cellulase system of Clostridium papyrosolvens C7 was fractionated by means of ion-exchange chromatography into at least seven high-molecular-weight multiprotein complexes, each with different enzymatic and structural properties. The molecular weights of the complexes, as determined by gel filtration chromatography, ranged from 500,000 to 660,000, and the isoelectric points ranged from 4.40 to 4.85. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the complexes showed that each complex had a distinct polypeptide composition. Avicelase, carboxymethyl cellulase, and xylanase activity profiles differed from protein complex to protein complex. Three of the complexes hydrolyzed crystalline cellulose (Avicel). Activity zymograms of gels (following electrophoresis under mildly denaturing conditions) revealed different carboxymethyl cellulase-active proteins in all complexes but xylanase-active proteins in only two of the complexes. The xylanase specific activity of these two complexes was more than eightfold higher than that of the unfractionated cellulase preparation. A 125,000-M(r) glycoprotein with no apparent enzyme activity was the only polypeptide present in all seven complexes. Experiments involving recombination of samples eluted from the ion-exchange chromatography column indicated that synergistic interactions occurred in the hydrolysis of crystalline cellulose by the cellulase system. We propose that the C. papyrosolvens enzyme system responsible for the hydrolysis of crystalline cellulose and xylan is a multicomplex system comprising at least seven diverse protein complexes.
TL;DR: A culture medium for increased xylanase formation by a wild strain of Sclerotium rolfsii was optimized by applying statistically designed experiments and resulted in elevated levels of other hemicellulolytic enzyme activities including mannanase, α-arabinosidase, and acetyl esterase.
Abstract: A culture medium for increased xylanase formation by a wild strain of Sclerotium rolfsii was optimized by applying statistically designed experiments. The optimization process was divided into three basic steps. In the initial phase of screening, two different fractional factorial plans, a Graeco-Latin square design and a folded Plackett-Burman design, were employed. From the list of medium components the relevant variables for xylanase formation (cellulose, peptone, and trace elements) were identified. The second step of optimization used a central composite experimental design to calculate a predictive model. In this phase only the two most important factors, i. e. cellulose and peptone from meat, were considered. The third step of verification validated the results of the optimization. Optimal concentrations of cellulose and peptone were found to be 42.6 and 80.0 g 1−1, respectively. Shaken flask cultivations of S. rolfsii using the optimized medium yielded a maximum xylanase activity of 394 IU ml−1 (6,570 nkat ml−1) within 13 days. Growth on the optimized medium also resulted in elevated levels of other hemicellulolytic enzyme activities including mannanase, α-arabinosidase, and acetyl esterase. Especially the value of 155 IU · ml−1 (2,580 nkat ml−1) for mannanase is remarkable since it appears that this is among the highest activities reported for fungal organisms.
TL;DR: A significant enhancing effect of α-glucuronidase on the hydrolysis of glucuronoxylan by pure xylanases was observed.
TL;DR: Two new structures, a tetrasaccharide and a pentasaccharides, both containing terminal 2-O-arabinofuranosylxylopyranose as a structural element are described.
TL;DR: Northern (RNA) blot analysis indicated that the xylanase T-6 transcript is about 1.4 kb and that the induction of this enzyme synthesis by xylose is on the transcriptional level.
Abstract: Bacillus stearothermophilus T-6 produces an extracellular thermostable xylanase. Affinity-purified polyclonal serum raised against the enzyme was used to screen a genomic library of B. stearothermophilus T-6 constructed in lambda-EMBL3. Two positive phages were isolated, both containing similar 13-kb inserts, and their lysates exhibited xylanase activity. A 3,696-bp SalI-BamHI fragment containing the xylanase gene was subcloned in Escherichia coli and subsequently sequenced. The open reading frame of xylanase T-6 consists of 1,236 bp. On the basis of sequence similarity, two possible -10 and -35 regions, a ribosome-binding site at the 5' end of the gene and a potential transcriptional termination motif at the 3' end of the gene, were identified. From the previously known N-terminal amino acid sequence of xylanase T-6 and the possible ribosome-binding site, a putative 28-amino-acid signal peptide was deduced. The mature xylanase T-6 consists of 379 amino acids with a calculated molecular weight and pI of 43,808 and 6.88, respectively. Multiple alignment of beta-glycanase amino acid sequences revealed highly conserved regions. Northern (RNA) blot analysis indicated that the xylanase T-6 transcript is about 1.4 kb and that the induction of this enzyme synthesis by xylose is on the transcriptional level.
TL;DR: The gene bgaA encoding an alkaline endo-beta-1-3-1,4-glucanase (lichenase) from an alkalophilic Bacillus sp.
Abstract: The gene xyaA encoding an alkaline endo-beta 1,4-xylanase from an alkalophilic Bacillus sp. strain (N137) isolated in our laboratory was cloned and expressed in Escherichia coli. The nucleotide sequence of a 1,656-bp DNA fragment containing xyaA was determined, revealing one open reading frame of 993 bp that encodes a xylanase (XyaA) of 39 kDa. This xylanase lacks a typical putative signal peptide, yet the protein is found in the Bacillus culture supernatant. In Escherichia coli, the active protein is located mainly in the periplasmic space. The xylanase activity of the cloned XyaA is an endo-acting enzyme that shows optimal activity at pH 8 and 40 degrees C. This activity is stable at a pH between 6 and 11. Incubations of XyaA at 40 degrees C for 1 h destroyed 45% of the activity.
TL;DR: The two genes of B. stearothermophilus 21 appear to be, at least partly, expressed independently, which was experimentally confirmed in E. coli by deletion analysis.
Abstract: Bacillus stearothermophilus 21 is a gram-positive, facultative thermophilic aerobe that can utilize xylan as a sole source of carbon. We isolated this strain from soil, purified its extracellular xylanase and beta-xylosidase, and analyzed the two-step degradation of xylan by these enzymes (T. Nanmori, T. Watanabe, R. Shinke, A. Kohno, and Y. Kawamura, J. Bacteriol. 172:6669-6672, 1990). An Escherichia coli transformant carrying a 4.2-kbp chromosomal segment of this bacterium as a recombinant plasmid was isolated. It excreted active beta-xylosidase and xylanase into the culture medium. The plasmid was introduced into UV-sensitive E. coli CSR603, and its protein products were analyzed by the maxicell method. Proteins harboring beta-xylosidase and xylanase activities were identified, and their molecular masses were estimated by sodium dodecyl sulfate-polyarylamide gel electrophoresis to be 75 and 40 kDa, respectively. The values were identical to those of proteins prepared from cells of B. stearothermophilus 21. The genes for both enzymes were encoded in a 3.4-kbp PstI fragment derived from the 4.2-kbp chromosomal segment. The nucleotide sequence of the 4.2-kbp segment was accordingly determined. The beta-xylosidase gene (xylA) is located upstream of the xylanase gene (xynA) with a possible promoter and a Shine-Dalgarno sequence. The latter gene is preceded by two possible promoters and a Shine-Dalgarno sequence that are located within the 3'-terminal coding region of the former. The two genes thus appear to be, at least partly, expressed independently, which was experimentally confirmed in E. coli by deletion analysis.(ABSTRACT TRUNCATED AT 250 WORDS)
TL;DR: In this article, the pretreatment of softwood kraft pulps with endo-xylanase expressed from Streptomyces lividans (pIAF18) was shown to have beneficial effects on bleaching with selective oxygen-based bleaching reagents.
TL;DR: The enzyme induction was repressed by high concentration of glucose and this could not be reversed by exogenous cyclic-adenosine 3′,5′-monophosphate (cAMP).
TL;DR: Thermophilic Humicola sp.
TL;DR: Thermophilic (55° C) methanation of source-sorted household solid waste (HSW) was studied in batch and in continuous experiments and the methane yield by conversion of the HSW was not increased by enzyme additions (enzyme mixture).
Abstract: Thermophilic (55° C) methanation of source-sorted household solid waste (HSW) was studied in batch and in continuous experiments. Furthermore, the effects of additions of xylanase, lipase, protease and a mixture of these on the methanation were tested. In the batch studies, comparative assays with active and inactive enzymes were used to elucidate the role of the added enzymes. The results showed that the HSW was readily digestible, up to 400–590 mlCH4·g−1 volatile solids (VS) was produced. Only with protease added, at a concentration of 1.1 Anson protease units·kg−1 VS was a higher specific methanogenic activity found with active enzymes compared to inactive (autoclaved) enzymes or without enzyme addition. The methane yield by conversion of the HSW in the batch assays and in the reactor studies was not increased by enzyme additions (enzyme mixture).
TL;DR: The medium components for the production of extracellular xylanase by Thermoascus aurantiacus was optimized in shake-flask culture using the Box-Wilson method and a central composite design and the enzymes exhibited remarkable stabilities at high temperatures (50–70°C) for prolonged period.
TL;DR: In this article, the authors evaluated the effects of an experimental enzyme-inoculant mixture (264 ml/tonne of forage) containing cellulase, xylanase, cellobiase, and glucose oxidase activities on the course of corn silage fermentation.
TL;DR: Results indicate that the synthesis of xylanase, cellulase, and mannanase activities is likely to be under common regulatory control in the case of Schizophyllum commune, and the inducer seems to be a small molecule derived from cellulose.
TL;DR: A chimeric xylanase/endoglucanase with an internal cellulose-binding domain was constructed by fusing the Bacillus subtilis xyn gene fragment to the 5'-end of the Cellulomonas fimi cenA with a strong affinity for both microcrystalline cellulose (Avicel) and regenerated chitin.
Abstract: A chimeric xylanase/endoglucanase (XynCenA) with an internal cellulose-binding domain was constructed by fusing the Bacillus subtilis xyn gene fragment to the 5'-end of the Cellulomonas fimi cenA. A polyhistidine-encoding sequence was also fused to the 5'-end of the xyn gene. The gene fusion was overexpressed in Escherichia coli and the fusion polypeptide purified from the cell extracts using the polyhistidine tail. The hybrid protein behaved like the parental endoglucanase or xylanase when assayed on a number of soluble and insoluble cellulosic substrates or xylans. The presence of two distinct active sites and the internal cellulose-binding domain did not significantly affect the hydrolysis of any of these substrates. However, the fusion protein exhibited a strong affinity for both microcrystalline cellulose (Avicel) and regenerated chitin. Like the parental endoglucanase, bound XynCenA could not be eluted from these polysaccharides with either low or high salt buffer or distilled water. More stringent conditions, such as 1% SDS or 8 M guanidinium hydrochloride, fully desorbed the protein. The fusion protein did not adsorb significantly to insoluble xylan.
TL;DR: The xylanase A gene from Streptomyces lividans was modified by site-directed mutagenesis, selecting for mutations that improved the catalytic activity and thermostability of the enzyme.
TL;DR: Results indicated that the negative charge introduced by the mutation greatly affected the transglycosylation reaction catalysed by this xylanase, which affected the yield of hydrolysis products.
Abstract: The amino acid replacement of Asn173 by Asp in the xylanase A (Xln A) of Streptomyces lividans significantly altered its enzymic properties. A time-course hydrolysis of xylan showed that the altered xylanase ([N173D] Xln A) initially produced larger amounts of xylose (X1), xylobiose (X2) and xylotriose (X3) than Xln A, but less xylotetraose (X4). The bond-cleavage frequencies were determined for both enzymes using xylopentaose (X5), xylotetraose (X4) and xylotriose (X3) labelled at the reducing end of the molecule. Xln A hydrolysed X5, yielding 56% X2 and 44% X3, while [N173D]Xln A liberated 90% X2 and only 10% X3. Both enzymes hydrolysed X4 into 100% X2 and X3 into 100% X1. Transglycosylation reactions were detected in HPLC hydrolysis patterns using high substrate concentrations, where larger products than the starting substrates were formed. Their subsequent degradation also affected the yield of hydrolysis products. Using X5 as substrate, products from xylohexaose (X6) up to xylooligosides larger than xylooctaose (X8) were synthesized by Xln A, while [N173D]Xln A produced only a small amount of xyloheptaose (X7) and X8. Xln A hydrolysed X5 into an equivalent amount of X4 and X2 and 1.5-fold more X3. However, [N173D]Xln A yielded the same amount of X3 and X2 but half as much X4. With X4 as substrate, Xln A synthesized twofold more X7 and X6 than [N173D]Xln A. Xln A liberated 1.4-fold more X3 than X2, while [N173D]Xln A yielded twofold more X2 than X3. Xln A liberated almost fourfold more X2 than X1 from X3, while [N173D]Xln A produced only twofold more X2 than X1. These results indicated that the negative charge introduced by the mutation greatly affected the transglycosylation reactions catalysed by this xylanase.